The goal of this proposal is to investigate the motional changes that occur in response^[unreadable] protein kinase activation, and explore their relevance to enzyme function. Analysis of the MAP^kinase^ ERK2, by hydrogen exchange mass spectrometry (HX-MS) and other measurements reveal three situations in which enzyme perturbations involving (i) enzyme activation, (ii) allosteric communication between binding sites, and (iii) mutagenesis lead to localized changes in protein conformational mobility over long distances. The available evidence suggests a novel model for signal transduction control, in which the regulation of protein dynamics controls catalytic and allosteric functions in ERK2. Specific aims in this proposal will test this hypothesis, and examine behavior of related MAP kinases. Sp. Aim 1 will analyze protein dynamics in ERK2 by HX-MS, NMR and FRET in order to determine the functional consequence of flexibility changes at the hinge that are induced by kinase phosphorylation and activation. Sp. Aim 2 will test the hypothesis that MAP kinase docking motifs interact allosterically between their respective binding pockets, and explore the functional consequences of allosteric regulation, using isothermal calorimetry, HX-MS, and NMR. Sp. Aim 3 will investigate the mechanism by which mutations in the N-terminal domain of ERK2 regulate flexibility at the activation lip, by site-directed mutagenesis, HX-MS and NMR. Sp. Aim 4 will extend our HX-MS analyses to document activation-dependent conformational mobility in other protein kinases. Completion of these aims will provide a unique window for understanding how protein kinases have evolved to optimize function by controlling conformational mobility. Innovative technologies will be applied to this problem, including high field solution NMR and hydrogen exchange mass spectrometry. The experiments will: (i) document the regulation of internal motions in a large enzyme, (ii) demonstrate the role of protein motions in controlling kinase enzymatic function, (iii) improve our understanding of how protein motions are controlled over long distances, and (iv) document new mechanisms for regulation of signaling molecules.